Anti-pd-1/anti-her2 natural antibody structural heterodimeric bispecific antibody and method of preparing the same

ABSTRACT

Provided are an anti-PD-1/anti-HER2 natural antibody structural heterodimeric bispecific antibody and a method of preparing the same. Specifically, provided are a highly stable anti-PD-1/anti-HER2 heterodimeric bispecific antibody having characteristics of a natural IgG and having no mismatched heavy chain and light chain, and a method of preparing the same. The bispecific antibody may bind to both of two kinds of target molecules, and thus may be more effective in treating complex diseases.

TECHNICAL FIELD

The present disclosure relates to an anti-PD-1/anti-HER2 naturalantibody structural heterodimeric bispecific antibody and a method ofpreparing the same. Specifically, the present disclosure provides ahighly stable anti-PD-1/anti-HER2 heterodimeric bispecific antibodyhaving characteristics of a natural IgG and having no mismatched heavychain and light chain, and a method of preparing the same.

BACKGROUND ART

Monoclonal antibodies are highly specific antibodies that act only on asingle antigenic determinant and have been widely used for cancer,inflammation, autoimmune diseases, infectious diseases, etc. However,such therapeutic molecules do not exhibit sufficient pharmacologicaleffects when used alone. This is attributed to the complexity of thediseases. For example, cancer or inflammatory diseases are oftenassociated with interactions between molecular pathways and signalingpathways that mediate various diseases. In this case, a molecule havinga single target may not provide an optimal therapeutic effect, and thetherapeutic effect may be improved by simultaneously blocking moleculeslocated on multiple targets or at many sites on a single target. At thesame time, dual targeting therapy using multispecificity such asbispecific molecules may simplify the process of developing new drugsbecause a bispecific molecule is a single molecule. As compared withusing a combination of several monospecific molecules, this method is amore convenient method for both patients and healthcare providers.

Many different types of bispecific antibodies or bifunctional moleculeshave been known in the art. The first bispecific antibody was obtainedby coupling two IgG molecules, Fab′, or (Fab′)2 fragments using achemical method and a bifunctional coupling reagent. However, such achemically coupled bispecific antibody has many limitations, includingthe intensity of production work, purification of heterologous couplingproducts, complexity in the removal of homologous coupling products,original monospecific antibodies, or fragments, low efficiency, etc.

Another method used in the production of bispecific antibodies is to usea hybrid-hybridoma (quadroma) technology, which is a method of producinga bispecific antibody by somatic fusion of two kinds of hybridoma celllines that secrete different antibodies. Due to arbitrary pairing ofheavy and light chains of immunoglobulins, only one-tenth of theantibody mixture is the functional bispecific antibody required, thuscomplicating the purification process and reducing the production yield.

WO2013060867 describes a method of mass-producing a heterodimericbispecific antibody. In this method, a mixture of two kinds ofhomodimeric antibodies is first reduced, and asymmetric amino acidmutations are introduced into CH3 domains of the two kinds ofhomodimeric antibodies to promote Fab arm exchange of the differentantibodies, and inter-chain disulfide bonds of the hinge region areoxidized to form a stable bispecific antibody.

WO2009089004 describes a method of preparing a heterodimeric protein. Inthis method, amino acids at the CH3-CH3 interface are mutated intocharged amino acids such that heterodimerization is electrostaticallyfavorable but homodimer formation is electrostatically unfavorable.

U.S. Pat. No. 5,731,168 describes a method of preparing a heterodimericIgG using a protuberance-into-cavity strategy. In this method,“protuberances” are constructed by replacing small amino acids at theinterface of the CH3 domain of a first chain with larger amino acids,and at the same time, “cavities” are created by replacing correspondinglarge amino acids of the CH3 domain of a second chain with smaller aminoacids. The protuberance and cavity interaction is favorable toheterodimeric IgG formation but unfavorable to homodimer formation.

WO2012058768 describes a method of preparing a highly specific stableheterodimeric IgG. This method combines both negative and positivedesign strategies along with structural and computational modelingguided protein engineering techniques to mutate a plurality of aminoacids in the IgG1 CH3 structural domain, thereby forming a stableheterodimeric IgG with low homodimeric impurities.

Programmed death receptor-1 (PD-1) is an immune checkpoint that hasrecently attracted much attention, and is mainly involved in the controlof T cell activation, and regulates strength and duration of immuneresponses. Under normal conditions, PD-1 mediates and maintainsself-tolerance of body tissues and prevents damage of autologous tissuecaused by excessive activation of the immune system during aninflammatory process, and therefore, has a positive effect on theprevention of autoimmune diseases. Under pathological conditions, PD-1is involved in the occurrence and development of tumor immunity andvarious autoimmune diseases (Anticancer Agents Med Chem. 2015;15(3):307-13. Hematol Oncol Stem Cell Ther. 2014 March; 7(1):1-17.Trends Mol Med. 2015 January; 21(1):24-33. Immunity. 2013 Jul. 25;39(1):61-73. J Clin Oncol. 2015 Jun. 10; 33(17):1974-82.).

PD-1 belongs to the CD28 family, but unlike other members of the CD28family such as CTLA4, etc., it may form covalent dimers via disulfidebonds, and exists in a monomeric form. The structure of PD-1 mainlyincludes an extracellular immunoglobulin variable region as a structuraldomain, a hydrophobic transmembrane domain, and an intracellular domain.The intracellular domain includes two independent phosphorylation sites.The phosphorylation sites are an immunoreceptor tyrosine-basedinhibitory motif (ITIM) and an immunoreceptor tyrosine-based switchmotif (ITSM). PD-1 is inducibly expressed on the surface of activated Tcells, and also on B cells, NK cells, monocytes, and DC cells. PD-1ligands include programmed death ligand 1 (PD-L1) and programmed deathligand 2 (PD-L2), and these ligands belong to the B7 family. Of them,PD-L1 is inducibly expressed on the surface of various immune cellsincluding T cells, B cells, monocytes, macrophages, DC cells,endothelial cells, epidermal cells, etc., but PD-L2 is induciblyexpressed only on some immune cells such as macrophages, DC cells, Bcells, etc. (Autoimmun Rev, 2013, 12(11):1091-1100. Front Immunol, 2013,4:481. Nat Rev Cancer, 2012, 12(4): 252-264. Trends Mol Med. 2015January; 21(1): 24-33.).

In the 1980s, Dennis Slamon first discovered overexpression of the humanepidermal growth factor receptor 2 (HER2) gene in 30% of 189 primarybreast cancer cases, and revealed that HER2 is closely associated withoverall survival rate and recurrence time (Salman D J, et al., Science,235:177-182, 1985). According to recent studies, HER2 is overexpressedin approximately 25-30% of breast cancer patients (Revillion F et al.,Eur J Cancer, 34:791-808, 1998), which is associated with malignantprogression of tumors (Wright C et al., Cancer Res, 49: 2087-2090,1989).

Trastuzumab is a humanized monoclonal antibody against the HER2extracellular domain (Carter P et al., PNAS, 89(10):4285-4289, 1992).However, the anti-cancer effect of trastuzumab in clinical studies isoften lower than that in preclinical studies, and therefore, trastuzumabis generally used in combination with chemotherapeutic agents (Slamon DJ et al., N Engl J Med, 344:783-792, 2001).

Designing bi-functional antibodies capable of recruiting effector cellsis an effective means of improving the efficacy of antibodies. Untilnow, most studies have been conducted to exploit the function of the CD3molecule. A target tumor can be effectively removed through activationof killer T cells by the CD3 molecule (Haas C et al., Immunobiology,214:441-453, 2009). Among them, BiTE, which is a recombinantbifunctional T cell-stimulating antibody developed by Micromet, Inc.,has shown great promise, but the biggest problem is that its serumhalf-life is very short and its half-life in the human body is only 1hour (Loffler A et al., Blood, 95:2098-2103). This is attributed to thestructure of BiTE itself. BiTE is composed of two single-chain antibodyfragments. Its molecular weight is a mere 60 kDa, and Fc fragments whichplay an important role in half-life extension in an antibody moleculeare removed.

Catumaxomab is another type of promising multi-functional antibody, andis a hetero Ig molecule targeting CD3 and EpCAM. Currently, this productis approved for the treatment of malignant ascites (Jager M et al.,Cancer Res, 72:24-32, 2012). Still another multifunctional antibodyunder phase II clinical trial is ertumaxomab which targets CD3 and HER2.A heavy chain and a light chain of the heteroantibody is derived fromrat IgG and targets CD3; and another heavy chain and light chain isderived from mouse IgG and targets HER2. A problem which accompaniesthis is that production of these products is very difficult. To acquireclones expressing bifunctional ertumaxomab, one hybridoma expressing aCD3 antibody and one hybridoma expressing a HER2 antibody are firstprepared, respectively, and then the two hybridomas are hybridized toobtain a quadroma which is a bifunctional antibody capable of expressinganti-CD3 and HER2. Usually, in order to produce a single-targetantibody, only one hybridoma is needed. In comparison, the productionprocess of the bifunctional antibody is much more complicated, and it isalso more difficult to obtain the quadroma, which may result inextremely high immunogenicity due to its rat origin.

Further, the most obvious side effect of the anti-CD3 antibody is atransient burst of systemic cytokine release, also called a ‘cytokinestorm’. Accordingly, there is a demand for a new bifunctional antibodythat recruits immune cells to the surface of tumor cells.

DESCRIPTION OF EMBODIMENTS Technical Problem

A first aspect of the present disclosure relates to a heterodimericbispecific antibody including a first antigen-binding functional domaincapable of specifically binding to PD-1 and a second antigen-bindingfunctional domain capable of specifically binding to HER2. Thebispecific antibody may include a first Fc chain and a second Fc chainwhich are linked to each other via one or more disulfide bonds, whereinthe first Fc chain and the second Fc chain are respectively linked tothe PD-1 antigen-binding functional domain and the HER2 antigen-bindingfunctional domain, and the first Fc chain and the second Fc chaininclude 5 amino acid substitutions at the following positions:

1) amino acid substitutions at positions 366 and 399 of the first Fcchain and amino acid substitutions at positions 351, 407, and 409 of thesecond Fc chain; or

2) amino acid substitutions at positions 366 and 409 of the first Fcchain and amino acid substitutions at positions 351, 399, and 407 of thesecond Fc chain, wherein the first and second Fc chains including theabove amino acid substitutions respectively have a tendency to undergoheterodimerization rather than homodimerization, and the amino acidpositions are numbered according to the Kabat EU numbering system.

In an embodiment, the amino acid substitutions of the first Fc chain andthe second Fc chain are as follows:

a) substitution of glycine, tyrosine, valine, proline, aspartic acid,glutamic acid, lysine, or tryptophan at position 351;

b) substitution of leucine, proline, tryptophan, or valine at position366;

c) substitution of cysteine, asparagine, isoleucine, glycine, arginine,threonine, or alanine at position 399;

d) substitution of leucine, alanine, proline, phenylalanine, threonine,or histidine at position 407; and

e) substitution of cysteine, proline, serine, phenylalanine, valine,glutamine, or arginine at position 409.

In an embodiment, the amino acid substitutions may include:

a) T366L and D399R substitutions in the first Fc chain and L351E, Y407L,and K409V substitutions in the second Fc chain;

b) T366L and D399C substitutions in the first Fc chain and L351G, Y407L,and K409C substitutions in the second Fc chain;

c) T366L and D399C substitutions in the first Fc chain and L351Y, Y407A,and K409P substitutions in the second Fc chain;

d) T366P and D399N substitutions in the first Fc chain and L351V, Y407P,and K409S substitutions in the second Fc chain;

e) T366W and D399G substitutions in the first Fc chain and L351 D,Y407P, and K409S substitutions in the second Fc chain;

f) T366P and D399I substitutions in the first Fc chain and L351P, Y407F,and K409F substitutions in the second Fc chain;

g) T366V and D399T substitutions in the first Fc chain and L351K, Y407T,and K4090 substitutions in the second Fc chain;

h) T366L and D399A substitutions in the first Fc chain and L351W, Y407H,and K409R substitutions in the second Fc chain.

In an embodiment, the amino acid substitutions may include:

a) T366L and K409V substitutions in the first Fc chain and L351E, Y407L,and D399R substitutions in the second Fc chain;

b) T366L and K409C substitutions in the first Fc chain and L351G, Y407L,and D399C substitutions in the second Fc chain;

c) T366L and K409P substitutions in the first Fc chain and L351Y, Y407A,and D399C substitutions in the second Fc chain;

d) T366P and K409S substitutions in the first Fc chain and L351V, Y407P,and D399N substitutions in the second Fc chain;

e) T366W and K409S substitutions in the first Fc chain and L351 D,Y407P, and D399G substitutions in the second Fc chain;

f) T366P and K409F substitutions in the first Fc chain and L351P, Y407F,and D399I substitutions in the second Fc chain;

g) T366V and K4090 substitutions in the first Fc chain and L351K, Y407T,and D399T substitutions in the second Fc chain;

h) T366L and K409R substitutions in the first Fc chain and L351W, Y407H,and D399A substitutions in the second Fc chain.

In an embodiment, the first Fc chain has amino acid substitutions ofT366L and D399R, and the second Fc chain has amino acid substitutions ofL351E, Y407L, and K409V.

In an embodiment, the Fc chain is derived from IgG.

In an embodiment, the PD-1 and HER2 antigen-binding functional domainsare Fab fragments or scFv fragments.

In an embodiment, the PD-1 and HER2 antigen-binding functional domainsare all Fab fragments.

In an embodiment, one of the PD-1 and HER antigen-binding functionaldomains is a Fab fragment and the other is a scFv fragment.

In an embodiment, the Fab fragment may include a first heavy chainvariable region and a second heavy chain variable region that aredifferent form each other and a first light chain variable region and asecond light chain variable region that are different from each other.

In an embodiment, an amino acid sequence of the bispecific antibody isselected from SEQ ID NOs. 2, 4, 6, 8, 10, 12, 14, 16, and 18.

A second aspect of the present disclosure relates to an isolatedpolynucleotide encoding the heterodimeric bispecific antibody describedin the first aspect.

In an embodiment, a sequence of the polynucleotide is selected from SEQID NOs. 1, 3, 6, 7, 9, 13, 15, and 17.

A third aspect of the present disclosure relates to a recombinantplasmid including the isolated polynucleotide described in the secondaspect.

In an embodiment, the expression vector is a plasmid vector X0GC whichis obtained by modifying pcDNA.

A fourth aspect of the present disclosure relates to a host cellincluding the isolated polynucleotide described in the second aspect orthe recombinant expression vector described in the third aspect.

In an embodiment, the host cell is selected from a human embryonickidney cell HEK293, or HEK293T, HEK293F, or HEK293E derived from theHEK293 cell; and a Chinese hamster ovary cell CHO, or CHO-S, CHO-dhfr⁻,CHO/DG44, or ExpiCHO derived from CHO cell.

A fifth aspect of the present disclosure relates to a compositionincluding the heterodimeric bispecific antibody described in the firstaspect, the isolated polynucleotide described in the second aspect, therecombinant expression vector described in the third aspect, or the hostcell described in the fourth aspect, and a pharmaceutically acceptablecarrier.

A sixth aspect of the present disclosure relates to a method ofproducing the heterodimeric bispecific antibody described in the firstaspect, the method including:

1) expressing the isolated polynucleotide described in the second aspector the recombinant expression vector described in the third aspect in ahost cell;

2) reducing each protein expressed in the host cell; and

3) mixing the reduced proteins and then oxidizing the mixture.

In an embodiment, the host cell is selected from a human embryonickidney cell HEK293, or HEK293T, HEK293F, or HEK293E derived from theHEK293 cell; and a Chinese hamster ovary cell CHO, or CHO-S, CHO-dhfr⁻,CHO/DG44, or ExpiCHO derived from CHO cell.

In an embodiment, the reducing may include 1) adding a reducing agent,wherein the reducing agent is 2-mercaptoethylamine, dithiothreitol,tri(2-carboxyethyl)phosphine, other chemical derivatives, or acombination thereof, 2) performing a reduction reaction in the presenceof dithiothreitol at a concentration of 0.1 mM or more at 4° C. for atleast 3 hours, and 3) removing the reducing agent by desalting, etc.

In an embodiment, the oxidizing may include 1) oxidizing in air oradding an oxidizing agent, wherein the oxidizing agent is selected fromL-dehydroascorbic acid and other chemical derivatives, and 2) performingan oxidization reaction in the presence of L-dehydroascorbic acid at aconcentration of 0.5 mM or more at 4° C. for at least 5 hours.

In an embodiment, the method may further include isolating andpurifying.

A seventh aspect of the present disclosure relates to use of theheterodimeric bispecific antibody described in the first aspect, and/orthe isolated polynucleotide described in the second aspect, and/or therecombinant expression vector described in the third aspect, and/or thehost cell described in the fourth aspect, and/or the compositiondescribed in the fifth aspect, in the preparation of a drug forpreventing and/or treating a disease of a subject.

An eighth aspect of the present disclosure relates to use of theheterodimeric bispecific antibody described in the first aspect, and/orthe isolated polynucleotide described in the second aspect, and/or therecombinant expression vector described in the third aspect, and/or thehost cell described in the fourth aspect, and/or the compositiondescribed in the fifth aspect, in the prevention and/or treatment of adisease of a subject.

A ninth aspect of the present disclosure relates to a method ofpreventing and/or treating a disease, the method including administeringthe heterodimeric bispecific antibody described in the first aspect,and/or the isolated polynucleotide described in the second aspect,and/or the recombinant expression vector described in the third aspect,and/or the host cell described in the fourth aspect, and/or thecomposition described in the fifth aspect to a subject in need thereof.

In an embodiment, the subject may be a mammal, or preferably may be ahuman subject.

In an embodiment, the disease may be selected from tumors such asleukemia, lymphoma, myeloma, brain tumor, head and neck squamous cellcarcinoma, non-small cell lung cancer, nasopharyngeal carcinoma,esophageal cancer, stomach cancer, pancreatic cancer, gallbladdercancer, liver cancer, colon cancer, breast cancer, ovarian cancer,cervical cancer, endometrial cancer, uterine sarcoma, prostate cancer,bladder cancer, renal cell carcinoma, and melanoma.

Advantageous Effects of Disclosure

In the present disclosure, a completely new anti-PD-1/anti-HER2 naturalantibody structural heterodimeric bispecific antibody has been designedby employing PD-1 as a molecule recruiting immune cells and HER2 as atarget molecule of tumor cells. The bispecific antibody is a highlystable anti-PD-1/anti-HER2 heterodimeric bispecific antibody havingcharacteristics of a natural IgG and having no mismatched heavy chainand light chain. The bispecific antibody may bind to both of the twokinds of target molecules, PD-1 and HER-2, and thus may be moreeffective in treating complex diseases.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows an elution peak of a monomer of a heterodimeric antibodymolecule;

FIG. 2 illustrates a structure of an anti-PD-1/anti-HER2 heterodimericantibody molecule;

FIG. 3 illustrates a structure of a half-antibody molecule of a heavychain and a light chain;

FIG. 4 shows results of size-exclusion chromatography (SEC) analysis ofa half-antibody molecule of a heavy chain and a light chain, wherein Aand B show analysis results of an anti-HER2 half-antibody molecule andan anti-PD-1 half-antibody molecule, respectively;

FIG. 5 shows results of SDS-PAGE analysis of oxidized half-antibodymolecules of anti-PD-1 and anti-HER2 antibodies;

FIG. 6 shows an elution peak of the anti-PD-1/anti-HER2 heterodimericantibody molecule;

FIG. 7 shows results of SDS-PAGE analysis of the anti-PD-1/anti-HER2heterodimeric antibody molecule;

FIG. 8 shows results of SEC analysis of the anti-PD-1/anti-HER2heterodimeric antibody molecule;

FIG. 9 shows a stability test of the anti-PD-1/anti-HER2 heterodimericantibody molecule;

FIG. 10 shows a stability test of the anti-PD-1/anti-HER2 heterodimericantibody molecule;

FIG. 11 shows HER2-binding activity and PD-1-binding activity of theanti-PD-1/anti-HER2 heterodimeric antibody molecule, wherein A and Bshow HER2-binding activity and PD-1-binding activity, respectively;

FIG. 12 shows simultaneous binding of the anti-PD-1/anti-HER2heterodimeric antibody molecule to PD-1-overexpressing CHO/PD-1 cellsand HER2-overexpresing SK-BR-3 cells, wherein A to D show simultaneousbinding of a HER2 monoclonal antibody, a PD-1 monoclonal antibody, aHER2 monoclonal antibody+PD-1 monoclonal antibody, and anti-PD-1BJHM/anti-HER2, respectively;

FIG. 13 shows blocking activity of the anti-PD-1/anti-HER2 heterodimericantibody molecule against PD-1/PD-L1 binding and PD-1/PD-L2 binding,wherein A and B show blocking activity against PD-1/PD-L1 and PD-1/PD-L2binding, respectively;

FIG. 14 shows cytokine IL-2 secretion promoted by theanti-PD-1/anti-HER2 heterodimeric antibody molecule; and

FIG. 15 shows an area under curve (AUC) of the anti-PD-1/anti-HER2heterodimeric antibody molecule.

BEST MODE Definitions

Covalent linkage means that two Fc chains or any one Fc chain and anantigen-binding functional domain linked thereto in a heterodimericbispecific antibody are linked with each other via a covalent bond toform a single molecule. Among them, the Fc chain includes a firstantigen-binding functional domain and a second antigen-bindingfunctional domain linked via one or more covalent bonds (or disulfidebond chains); the first Fc chain and the second Fc chain each is linkedto one antigen-binding functional domain via a covalent bond (an iminebond or a peptide bond); and

The antigen-binding functional domain is a domain in which a specificinteraction with a target molecule such as an antigen occurs, and itsaction is highly selective, and a sequence that recognizes one targetmolecule generally does not recognize other molecule sequences. Arepresentative antigen-binding functional domain includes an antibodyvariable region, a structural variant of the antibody variable region, areceptor binding domain, a ligand binding domain, or an enzyme bindingdomain.

The linkage via one or more disulfide bond chains refers to formation ofa heterodimer fragment by a linkage between the first Fc chain and thesecond Fc chain via one or more disulfide bond chains. In the presentdisclosure, one or more disulfide bonds may be formed when the first Fcchain and the second Fc chain or the first Fc chain and the second Fcchain and the antigen-binding functional domains linked thereto aresynthesized in the same cell, or may be formed by in vitro reductionafter the first Fc chain and the second Fc chain or the first Fc chainand the second Fc chain and the antigen-binding functional domainslinked thereto are synthesized in the different cells, respectively.

The first Fc chain and the second Fc chain constitutes a bindingfragment via a covalent bond, and the covalent bond include a disulfidebond, and each chain includes at least a part of an immunoglobulin heavychain constant region; the first chain and the second chain aredifferent from each other in their amino acid sequences and include adifferent amino acid at least one position. In the first Fc chain andthe second Fc chain of the present disclosure, a strong repulsive forceexists between the same chains and an attractive force exists betweenthe different chains. Therefore, the first Fc chain and the second Fcchain or the first Fc chain and the second Fc chain and theantigen-binding functional domains linked thereto have a tendency toundergo heterodimeric formation, when co-expressed in a cell. When thefirst Fc chain and the second Fc chain or the first Fc chain and thesecond Fc chain and the antigen-binding functional domains linkedthereto are expressed in different two host cells, respectively, thefirst Fc chains, or the first Fc chain and the antigen-bindingfunctional domain linked thereto have no tendency to undergo homodimericformation, and the second Fc chains, or the second Fc chain and theantigen-binding functional domain linked thereto also have no tendencyto undergo homodimeric formation. In the present disclosure, when thefirst Fc chain and the second Fc chain, or the first Fc chain and thesecond Fc chain and the antigen-binding functional domains linkedthereto are expressed in two different host cells, respectively and areducing agent is present, a percentage of homodimers is less than 50%,that is, a percentage of monomers (one Fc chain or one Fc chain and oneantigen-binding functional domain linked thereto) is 50% or more.

An immunoglobulin has a symmetric structure having four polypeptidechains; two chains are identical heavy chains which are relatively longand have a relatively high molecular weight, each including 450 to 550amino acid residues and having a relative molecular weight of 55000 Dato 70000 Da; and the other two chains are identical light chains (Lchains) which are relatively short and have a relatively low molecularweight, each including 210 amino acid residues and having a relativemolecular weight of about 24000 Da. About 110 amino acid sequences nearthe N-terminus of immunoglobulin heavy and light chains are highlyvariable, and the region is called a variable region (V region), and therest amino acid sequences near the C-terminus thereof are relativelystable, called a constant region (C region). The variable region in theheavy chain occupies approximately ¼ of the length of the heavy chain,and the constant region occupies approximately ¾ of the length of theheavy chain. The known 5 types of immunoglobulins are IgG(γ), IgA(α),IgD(δ), IgM(μ) and IgE(ε). Among them, the former three kinds ofimmunoglobulins have three constant regions consisting of CH1, CH2 andC3 in H chain, and the latter two kinds of immunoglobulins (IgM and IgE)have one VH domain and four constant domains, i.e., CH1 to CH4 in Hchain. The constant region is a framework of an immunoglobulin molecule,and is also one of regions activating immune responses.

A part of the constant region of the present disclosure includes atleast an interaction region of the first Fc chain and the second Fcchain, and in the case of IgG, this region is located at any amino acidpositions of CH3 domain and includes at least GLN347, TYR349, THR 350,LEU 351, SER 354, ARG 355, ASP 356, GLU 357, LYS 360, SER 364, THR 366,LEU 368, LYS 370, ASN390, LYS392, THR394, PRO395, VAL 397, ASP399,SER400, PHE405, TYR407, LYS409, LYS439.

The first Fc chain and the second Fc chain each linked to oneantigen-binding functional domain via a covalent bond or a linkerindicate the first Fc chain and the second Fc chain each linked to anantigen-binding fragment of one antibody, or a single chain antibodycapable of recognizing an antigen, or other antibody fragment variantcapable of recognizing an antigen, or a receptor capable of recognizinga ligand, or a ligand capable of recognizing a receptor via a covalentbond or a linker. The covalent bond is a kind of chemical bonding, inwhich two or more atoms together use their outer electrons, in the idealcase of electronic saturation, thus forming a relatively stable chemicalstructure, or the interaction between atoms is formed by shared electronpair. Atoms of the same element or atoms of different elements may beall linked via the covalent bond. The covalent bond of the first Fcchain and the second Fc chain of the present disclosure may include anamide bond formed by dehydration between an amino group of an amino acidof one molecule and a carboxyl group of an amino acid of anothermolecule, or an amide bond between an aldehyde group of ethylene glycolor polyethylene glycol or other compound or a polymer thereof and anamino group of an amino acid of one molecule, but is not limitedthereto. The linker is one amino acid sequence or one compound or onemultimer of the one compound capable of linking two polypeptide chainsvia a covalent bond. Among them, the one amino acid sequence mayinclude, but is not limited to, a small peptide, such asGGGGSGGGGSGGGGS, and the amino acid sequence may link the first Fc chainor the second Fc chain and a single chain antibody capable ofrecognizing an antigen or other antibody fragment structural variantcapable of recognizing an antigen via an amide bond.

The first Fc chain and the second Fc chain have a tendency to undergoheterodimeric formation and no tendency to undergo homodimericformation, which means that in the first Fc chain and the second Fcchain, a strong repulsive force exists between the same polypeptidechains and an attractive force exists between the different polypeptidechains, and therefore, the first Fc chain and the second Fc chain or thefirst Fc chain and the second Fc chain and the antigen-bindingfunctional domains linked thereto have a tendency to undergoheterodimeric formation, when co-expressed in a cell. When the first Fcchain and the second Fc chain or the first Fc chain and the second Fcchain and the antigen-binding functional domains linked thereto areexpressed in two different host cells, respectively, the first Fcchains, or the first Fc chain and the antigen-binding functional domainlinked thereto have no tendency to undergo homodimeric formation, andthe second Fc chains, or the second Fc chain and the antigen-bindingfunctional domain linked thereto also have no tendency to undergohomodimeric formation.

The Kabat EU numbering system means that Kabat assigns a number to eachamino acid in an antibody sequence, and this method of assigning residuenumbers has become standard in the field. The Kabat's method isextendible to other antibodies not included in his compendium byaligning a target antibody with one of the consensus sequences in Kabatby reference to conserved amino acids.

Fc fragment region refers to a fragment crystallizable (Fc) andcorresponds to CH2 and CH3 structural domains of Ig, and is a fragmentwhere an interaction between Ig and an effector molecule or a celloccurs.

IgG is an abbreviation for immunoglobulin G (IgG), and is the main typeof antibody in the serum. Human IgG has four subclasses of IgG1, IgG2,IgG3, and IgG4 based on antigenic differences in r chains in the IgGmolecule.

A half antibody molecule has a structure formed by one heavy chain andone light chain of an antibody, wherein the heavy chain and the lightchain may be linked via a covalent bond, or has a monovalent antibodystructure recognizing an antigen, which may be formed without a covalentbond.

Fab fragment is a molecule-recognizing sequence, and a fragment ofantigen binding (Fab), and corresponds to two arms of an antibodymolecule, each consisting of a complete light chain and VH and CH1structural domains of a heavy chain. scFv is a molecule-recognizingsequence, and is a structural isomer of an antibody fragment obtained bygenetic modification of a light chain variable region and a heavy chainvariable region of an antibody. An extracellular domain of a membranereceptor is a molecule-recognizing sequence, and the membrane receptorusually includes an extracellular region that is located outside thecell and recognizes and binds to the corresponding antigen or ligand, atransmembrane region that anchors the receptor onto the cell surface,and an intracellular region that has intracellular kinase activity or asignaling pathway. The ligand of the cell membrane receptor refers to aprotein, a small peptide, or a compound that may be recognized and boundby the extracellular region of the membrane receptor. Cytokines arelow-molecular weight soluble proteins that are produced by various typesof cells induced by immunogens, mitogens, or other stimulants, and havevarious functions such as innate immunity and adaptive immunity,hematopoiesis, cell growth, APSC multifunctional cell and damage tissuerepair, etc. Cytokines may be classified into interleukins, interferons,tumor necrosis factor superfamilies, colony stimulating factors,chemotactic factors (chemokines), growth factors, etc. A proteinexpression tag means an amino acid sequence added at the N-terminus orC-terminus of a target protein, and may be small peptides or long aminoacids. Addition of the tag may be advantageous for correct folding ofproteins, protein isolation and purification, and intracellular proteindegradation. Tags frequently used may include HA, SUMO, His, GST, GFP,and Flag, but are not limited thereto.

There is no limitation in the antibodies applicable to the heterodimericbispecific antibody of the present disclosure. The antibodies alreadyused in the art for the treatment and/or prevention of diseases may beapplied to the present disclosure.

The heterodimeric bispecific antibody of the present disclosure may haveone or more substitutions, deletions, additions, and/or insertions. Forexample, although some amino acids are substituted for the amino acidsin the structure of the protein, there is no significant loss of abilityto bind to other polypeptides (e.g., antigens) or cells. Since thebinding ability and properties of the protein determine the biologicalfunctional activity of the protein, substitution of some amino acids onthe protein sequence may cause no significant loss of its biologicalusefulness or activity.

In many cases, polypeptide variants may include one or more conservativesubstitutions. The “conservative substitution” means that amino acids inthe polypeptide variants are replaced by other amino acids havingsimilar properties, such that one skilled in the art of peptidechemistry would expect a secondary structure and hydrophilic nature ofthe polypeptide to be substantially unchanged.

Amino acid substitutions may be generally based on relative similarityof amino acid side-chain substituents such as hydrophobicity,hydrophilicity, charge, size, etc. Exemplary substitutions that takevarious characteristics described above into consideration are wellknown to those skilled in the art and include arginine and lysine;glutamic acid and aspartic acid; serine and threonine; glutamine andasparagine; and valine, leucine, and isoleucine.

As used herein, the term “identity” has the meaning commonly known inthe art, and those skilled in the art also are familiar with the rulesand criteria for determining identity between different sequences, andthe identity refers to the percentage of homology between residues of apolynucleotide or polypeptide sequence variant and residues of anon-variant sequence after aligning the sequences and introducing gaps(if necessary, to achieve the maximum % homology). In the presentdisclosure, when the definition of identity is satisfied, it is alsorequired that the obtained variant sequence has the biological activitypossessed by the parent sequence. Methods and means for screeningvariant sequences using the above activities are well known to thoseskilled in the art. Such variant sequences may be readily obtained bythose skilled in the art from the teachings herein. In a specificembodiment, the polynucleotide and polypeptide variants have at leastabout 70%, at least about 75%, at least about 80%, at least about 90%,at least about 95%, at least about 98%, or at least about 99%, or atleast about 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or99.9% polynucleotide or polypeptide identity with the polynucleotide orpolypeptide described herein. Due to redundancy of the genetic code,variants of these sequences encoding the same amino acid sequence willexist.

Another aspect provides a polynucleotide composition capable ofhybridizing to the polynucleotide sequence provided by the presentdisclosure or a fragment thereof or a complementary sequence thereofunder moderately to highly stringent conditions. Hybridizationtechniques are well known in the art of molecular biology. For thepurposes of explanation, suitable moderately stringent conditions fortesting hybridization of the polynucleotide of the present disclosure toanother polynucleotide may include pre-washing with a solution of 5×SSC,0.5% SDS, 1.0 mM EDTA (pH 8.0); performing hybridization in 5×SSC at 50°C. to 60° C. overnight; and washing twice with 2×, 0.5× and 0.2×SSCcontaining 0.1% SDS for 20 minutes at 65° C., respectively. Thoseskilled in the art understand that the stringency of hybridization maybe readily manipulated, for example, by varying the salt content of thehybridization solution and/or the hybridization temperature. Forexample, in another embodiment, suitable highly stringent hybridizationconditions may include the conditions described above, except forincreasing the hybridization temperature, for example, to 60° C. to 65°C. or 65° C. to 70° C.

The host cell of the present disclosure may be any cell which may beused in foreign gene expression, and may include E. coli, yeast cells,insect cells, plant cells, and mammalian cells, but is not limitedthereto.

The vector of the present disclosure may be a vector which may replicatein any type of cells or organisms, and may include, for example,plasmids, bacteriophages, cosmids, and minichromosomes. In anembodiment, the vector including the polynucleotide of the presentdisclosure is a vector suitable for propagation or replication of apolynucleotide, or a vector suitable for expression of the polypeptideof the present disclosure. Such vectors are known in the art and arecommercially available.

The “vector” may include a shuttle vector and an expression vector.Generally, a plasmid construct may also include an origin of replication(e.g., ColE1 origin of replication) and a selectable marker (e.g.,ampicillin or tetracycline resistance) which are for plasmid replicationand selection in bacteria, respectively. The “expression vector” refersto a vector including a control sequence or a regulatory element whichis required for expression of the antibody of the present disclosure,including antibody fragments, in bacterial or eukaryotic cells.

The vector of the present disclosure may be any vector used for foreigngene expression, and may include, but is not limited to, a plasmidvector, wherein the plasmid vector includes at least an origin ofreplication, a promoter, a gene of interest, a multiple cloning site, aselection marker gene, and the vector of the present disclosure mayinclude, but is not limited to, a plasmid vector obtained based onpcDNA, such as X0GC vector.

The subject of the present disclosure may include birds, reptiles,mammals, etc. The mammal may include a rodent, a primate. The primatemay include a human.

The scope of the diseases involved in the present disclosure mayinclude, but is not limited to, tumors. The tumors may include leukemia,lymphoma, myeloma, brain tumor, head and neck squamous cell carcinoma,non-small cell lung cancer, nasopharyngeal carcinoma, esophageal cancer,stomach cancer, pancreatic cancer, gallbladder cancer, liver cancer,colon cancer, breast cancer, ovarian cancer, cervical cancer,endometrial cancer, uterine sarcoma, prostate cancer, renal cellcarcinoma, melanoma.

The pharmaceutically acceptable carrier means a pharmaceutical carrierwhich is commonly used in the pharmaceutical art, for example, diluents,excipients, water, etc., fillers such as starch, sucrose, lactose,microcrystalline cellulose, etc.; binders such as cellulose derivatives,alginates, gelatin and polyvinylpyrrolidone; wetting agents such asglycerin; disintegrating agents such as sodium carboxymethyl starch,hydroxypropyl cellulose, croscarmellose, agar, calcium carbonate, sodiumhydrogencarbonate, etc.; absorption enhancers such as quaternaryammonium compounds; surfactants such as cetanol, sodium lauryl sulfate,etc.; adsorption carriers such as kaolinite, bentonite, etc.; lubricantssuch as talc, calcium and magnesium stearate, micronized silica gel,polyethylene glycol, etc. In addition, other additives such as flavoringagents, sweeteners, etc. may be added to the composition.

MODE OF DISCLOSURE

Hereinafter, the present disclosure will be described in more detailwith reference to the following non-limiting examples. It will beunderstood by those skilled in the art that various modifications andchanges may be made therein without departing from the spirit and scopeof the present disclosure, and the modifications and changes are alsoincluded in the scope of the present disclosure.

The following experimental methods are all common methods unlessotherwise specified, and the experimental materials used may be alsoeasily obtained from commercial companies unless otherwise specified.The various antibodies used in the following Examples of the presentdisclosure are all standard antibodies obtained from the commercialroute.

Example 1: Construction of Vector Structure of Anti-PD-1/Anti-HER2Heterodimeric Antibody Molecule

X0GC expression vectors including heavy chain and light chain of humananti-PD-1(Pem) antibody were constructed, respectively. A sequence ofthe variable region of the antibody is derived fromhttp://www.imgt.org/3Dstructure-DB/cgi/details.cgi?pdbcode=9798. Anucleotide sequence of the light chain variable region is the same as inSEQ ID NO. 9 and an amino acid sequence thereof is the same as in SEQ IDNO. 10; a nucleotide sequence of the light chain constant region is thesame as in SEQ ID NO. 3 and an amino acid sequence thereof is the sameas in SEQ ID NO. 4; a nucleotide sequence of the heavy chain variableregion is the same as in SEQ ID NO. 11 and an amino acid sequencethereof is the same as in SEQ ID NO. 12; a nucleotide sequence of theheavy chain constant region is the same as in SEQ ID NO. 13 and an aminoacid sequence thereof is the same as in SEQ ID NO. 14. The light chainvariable region and the light chain constant region, and the heavy chainvariable region and the heavy chain constant region were amplified byPCR, respectively. In all PCR reactions of the present disclosure,Phusion high-fidelity DNA polymerase (F-530L) of NEB, Inc. was used. PCRprimers were designed commonly according to the principle of basecomplementation and the need for restriction enzyme sites. The reactionconditions consisted of 8.9 μl of H₂O, 4 μl of 5× Phusion high-fidelityDNA polymerase buffer, 4 μl of 1 mM dNTP, 1 μl of forward primer, 1 μlof reverse primer, 0.1 μl of Phusion high-fidelity DNA polymerase, and 1μl of the template. PCR products of the variable region and the constantregion were electrophoresed on 1.5% agarose gel, and fragmentscorresponding thereto were recovered using a DNA recovery kit (Promega,A9282, the same applies hereinafter). The recovered variable regionfragment and constant region fragment were used as templates and aforward primer of the variable region and a reverse primer of theconstant region were used to perform PCR. Fragments correspondingthereto were recovered to obtain a full length fragment of the heavychain and the light chain. XOGC vector and the full length fragment weredigested with EcoRI (NEB, Cat. No. R3101L) and HindiII (NEB, Cat. No.R3104L). The enzyme restriction conditions consisted of 2 μl of 10×buffer 3, each 0.5 μl of EcoRI and HindIII, 3 μl of full length fragmentrecovered from the gel, and 14.5 μl of H₂O. The restriction enzymes wereallowed to react at 37° C. for three hours. The restriction productswere ligated using T4DNA ligase (NEB, Cat. No. M0202V) (the same applieshereinafter), and the reaction conditions consisted of 2 μl of 10×ligase buffer, 0.5 μl of ligase, 3 μl of the full length fragmentrecovered from the gel, 3 μl of the XOGC vector recovered from the gel,and 11.5 μl of H₂O, which were ligated at room temperature for 12 hours.The ligation product was transformed into E. coli competent cell DH5a(Tiangen, CB104, the same applies hereinafter). The XOGC expressionvectors of antibody heavy chain and light chain were obtained in orderto express the antibody heavy chain and light chain in eukaryotic cells,respectively.

In the present disclosure, another XOGC expression vectors includingheavy chain and light chain of anti-human PD-1(BJHM) antibody were alsoconstructed, respectively. A nucleotide sequence of the light chainvariable region is the same as in SEQ ID NO. 15 and an amino acidsequence thereof is the same as in SEQ ID NO. 16; a nucleotide sequenceof the light chain constant region is the same as in SEQ ID NO. 3 and anamino acid sequence thereof is the same as in SEQ ID NO. 4; a nucleotidesequence of the heavy chain variable region is the same as in SEQ ID NO.17 and an amino acid sequence thereof is the same as in SEQ ID NO. 18; anucleotide sequence of the heavy chain constant region is the same as inSEQ ID NO. 13 and an amino acid sequence thereof is the same as in SEQID NO. 14. The XOGC expression vectors of antibody heavy chain and lightchain were obtained in order to express the antibody heavy chain andlight chain in eukaryotic cells, respectively.

In the present disclosure, XOGC expression vectors including heavy chainand light chain of anti-human HER2 antibody were also constructed,respectively. A sequence of the antibody variable region is derived fromhttp://www.drugbank.ca/drugs/DB00072, and the heavy chain constantregion is human IgG1(Fc2). A nucleotide sequence of the light chainvariable region is the same as in SEQ ID NO. 1 and an amino acidsequence thereof is the same as in SEQ ID NO. 2; a nucleotide sequenceof the light chain constant region is the same as in SEQ ID NO. 3 and anamino acid sequence thereof is the same as in SEQ ID NO. 4; a nucleotidesequence of the heavy chain variable region is the same as in SEQ ID NO.5 and an amino acid sequence thereof is the same as in SEQ ID NO. 6; anucleotide sequence of the heavy chain constant region is the same as inSEQ ID NO. 7 and an amino acid sequence thereof is the same as in SEQ IDNO. 8. The XOGC expression vectors of antibody heavy chain and lightchain were obtained in order to express the antibody heavy chain andlight chain in eukaryotic cells, respectively.

Example 2: Expression of Anti-PD-1/Anti-HER2 Heterodimeric AntibodyMolecule

The expression vectors including the heavy chain and the light chain ofanti-human PD-1 antibody were transfected into 293F cells (FreeStyle™293-F Cells, Cat. No. R79007, invitrogen), respectively, and theexpression vectors including the heavy chain and the light chain ofanti-human HER2 antibody were also transfected into 293F cells,respectively. One day before transfection, cells were seeded. On the dayof transfection, cells were collected by centrifugation, and resuspendedin fresh FreeStyle™ 293 expression medium (Cat. No. 12338001, Gibco) ata cell density of 200*10⁵ cells/mL. The plasmid was added according tothe transfection volume, and when a final concentration was 36.67 μg/mL,homogeneous mixing was lightly performed. Next, linear polyethyleneimine (PEI, linear, M.W. 25000, Cat. No. 43896, Alfa Aesar) was added,and when a final concentration was 55 μg/mL, homogeneous mixing waslightly performed. Next, the mixture was placed in an incubator, andincubated under shaking at a speed of 120 rpm at 37° C. for 1 hour.Next, 19 times transfection volume of fresh medium was added thereto.Incubation was continuously performed under shaking at a speed of 120rpm at 37° C. Culture supernatants of the transfected cells incubatedfor 5 to 6 days were collected by centrifugation.

Expression levels were determined by ELISA. Before purification byapplying the culture supernatant to a chromatography column, theprecipitate was removed by filtering through a 0.2 μm filter. Thisprocedure was performed at 4° C.

Example 3. Purification of Anti-PD-1/Anti-HER2 Heterodimeric AntibodyMolecule Expression Product

Purification was performed at 4° C. using an AKTA explorer 100 typeprotein purification system (GE Healthcare) and affinity chromatographyrProtein A Sepharose Fast Flow (16 mm I.D., 22 ml, GE Healthcare).First, a mobile phase A (20 mM sodium phosphate buffer, 150 mM sodiumchloride, pH 7.4) was used to equilibrate the chromatography column.After a baseline was stabilized, the supernatant of the above treatedcells was loaded at a flow rate of 5 mL/min. After loading the sample,equilibration was performed using the mobile phase A. The sample was theanti-PD-1 expression product and the anti-HER2 expression product,respectively. Thereafter, a mobile phase B1 (mobile phase A containing0.5 M arginine) was used to elute 5 column volumes; 5 column volumeswere washed with a mobile phase B2 (100 mM citric acid, pH 3.0) tocollect an elution peak, i.e., a peak of the protein of interest; a flowrate during the washing was all 5 ml/min. A chromatogram of the elutionpeak of anti-PD-1-Fc1 is as shown in FIG. 1, and an elution peak ofanti-HER2-Fc2 was also similar thereto (result is not shown). Theindicated elution peak (grey area shown) was collected and pH wasadjusted to 5.0 by dropwise addition of 1 M sodium acetate solution.

Example 4. Purification of Anti-PD-1/Anti-HER2 Heterodimeric AntibodyMolecule

The structure of the anti-PD-1/anti-HER2 heterodimeric antibody moleculeis as illustrated in FIG. 2.

The anti-PD-1 and anti-HER2 expression products obtained by theabove-described rProtein A Sepharose Fast Flow(16 mm I.D., 22 ml, GEHealthcare) method were subjected to in vitro recombination to obtain aheterodimer. First, the protein solutions purified and collected wereconcentrated by ultrafiltration through an ultrafiltration concentratingtube (nominal molecular weight cut-off of 10 kDa), and the solution wasreplaced by phosphate buffer saline (PBS) (pH=7.4). The obtainedanti-PD-1 and anti-HER2 expression products were adjusted to 1 mg/mlwith addition of PBS, and 1/200 times the final volume of 1 M DTT wasadded such that the final concentration of DTT was 5 mM, respectively.The reduction was carried out at 4° C. (3 hours to 8 hours), and thedisulfide bonds were opened through the reduction process, and thedisulfide bonds of the hinge region of a small amount of antibodyhomodimer molecules contained in the anti-PD-1 and anti-HER2 expressionproducts were also opened, thereby forming a half-antibody moleculecontaining one heavy chain and one light chain, of which structure is asillustrated in FIG. 3. The reduced sample was analyzed by SEC-HPLCcontaining 1 mM DTT reducing agent in the mobile phase buffer. Theresults are as shown in FIG. 4. A weight ratio of anti-PD-1 andanti-HER2 homodimers was all less than 10%. Consistent therewith, aweight ratio of the half antibody molecules was all more than 90%.

Thereafter, the reduced anti-PD-1 and anti-HER2 half antibody moleculeswere mixed according to a molar ratio, and recombination reaction wasallowed at 4° C. for 24 hours. During recombination, a heterodimericbispecific antibody including both the anti-PD-1 and anti-HER2 halfantibody molecules was formed via non-covalent interaction between CH2and CH3 of the anti-PD-1 and anti-HER2 half antibody molecules. Then,the protein solution was concentrated by ultrafiltration through anultrafiltration concentrating tube (nominal molecular weight cut-off of10 kDa), and the solution was replaced by PBS (pH=7.4) to terminate thereduction. The solution was subjected to oxidation in the air or with anoxidizing agent to allow formation of disulfide bonds of theheterodimeric bispecific antibody. The oxidation conditions were asfollows. 100 mM L-dehydroascorbic acid as the oxidizing agent was added,and when the final concentration of the protein became 1 mg/ml and thefinal concentration of the oxidizing agent became 1 mM, oxidation wasperformed at 4° C. for 24 hours. A sample obtained by theabove-described oxidation was subjected to SDS-PAGE analysis, and theresults are as shown in FIG. 5.

The heterodimer molecules obtained by the above-describedreduction/oxidation of the anti-PD-1 and anti-HER2 half antibodymolecules were concentrated by ultrafiltration through anultrafiltration concentrating tube (nominal molecular weight cut-off of10 kDa), and the solution was replaced by a sodium phosphate buffersolution (pH=5.8). Purification was performed at 4° C. using an AKTAexplorer 100 type protein purification system (GE Healthcare) and ionchromatography column Source 15S (16 mm I.D., 17 ml, GE Healthcare).First, a mobile phase A (10 mM sodium phosphate, pH 7.0) was used toequilibrate the chromatography column. After a baseline was stabilized,the above-treated protein solution was loaded at a flow rate of 3ml/min. After loading the sample, equilibration was performed using themobile phase A. Thereafter, 20 column volumes (0% B-100% B, 170 min,flow rate 2 ml/min) were washed with a gradient of A (10 mM sodiumphosphate, pH 5.8) to B (10 mM sodium phosphate, pH 5.8). The indicatedelution main peak was collected (see FIG. 6), and the collected proteinsolution was concentrated by ultrafiltration through an ultrafiltrationconcentrating tube (nominal molecular weight cut-off of 10 KDa). Thesolution was replaced by a phosphate solution (PBS, pH=7.4), andfiltered and sterilized, and then stored at 4° C. The purified productwas analyzed by SDS-PAGE method, and the results are as shown in FIG. 7.As a result of purity analysis by SEC-HPLC, the purity was 97.3%, asshown in FIG. 8.

Example 5. Stability of Anti-PD-1/Anti-HER2 Heterodimeric AntibodyMolecule

10 mg/mL of the PD-1/HER2 heterodimer samples fully sealed was left in aconstant climate chamber (BINDER KBF240) at 40° C., and 20 μg of thesample was taken at corresponding time points (baseline (Day 0), after 1day, 3 days, 5 days, 7 days and 14 days) and separated by size exclusionchromatography (SEC-HPLC). The above SEC-HPLC conditions were asfollows: (1) Size exclusion chromatography column: TSKgel G3000SWxI(Tosoh Bioscience), 5 μm, 7.8 mm×30 cm; (2) Mobile phase: 5 mM PBS, 150mM NaCl, pH 6.7; (3) Flow rate: 0.6 mL/min; (4) UV detection wavelength:280 nm; (5) Collection time: 30 min. The instrument used was an Agilent1200 Infinity chromatography, and chromatograph was recorded using anAgilent ChemStation and a ratio of remaining monomers was calculated. Asshown in FIGS. 9 (10 mg/mL) and 10 (1 mg/mL), the dimer did not undergosignificant aggregation under the experimental conditions of 40° C., andtherefore, the PD-1/HER2 heterodimer was considered to have excellentthermal stability.

Example 6. In Vitro Binding Activity of Anti-PD-1/Anti-HER2Heterodimeric Antibody Molecule

The binding ability of the PD-1/HER2 heterodimer antibody to a singleantigen was determined by enzyme-linked immunosorbent assay (ELISA).

Detailed procedures are as follows: Recombinant human PD-1 (BeijingYiqiao Shenzhou, Cat. No. 10377-H08H) or human HER2 (Beijing Yiyi) wascoated on a 96-well high adsorption ELISA plate using a carbonate buffersolution of pH 9.6 at a coating concentration of 1 μg/mL and a coatingamount of 100 μL per well. The coating was performed at 4° C. overnight.The plate was washed with PBST five times. The plate was blocked with300 μL/well of PBST containing 1% BSA and incubated for 1 hour at 25°C., and washed with PBST five times. A heterodimeric antibody sample anda control each serially diluted with PBST containing 1% BSA were addedin an amount of 100 μL per well, and incubated at 25° C. for 1 hour. Theplate was washed with PBST five times. Then, horseradishperoxidase-labeled anti-human IgG antibody (Chemicon, Cat. No. AP309P)diluted 1:2000 with PBST containing 1% BSA was added in an amount of 100μL per well, and incubated at 25° C. for 1 hour. The plate was washedwith PBST five times. A colorimetric substrate TMB was added in anamount of 100 μL/well and developed for 10 minutes at room temperature.Color development was terminated by adding 100 μL/well of 1 M H₂SO₄. Theabsorbance at 450 nm was read on a microplate reader.

As a result, as shown in FIG. 11, anti-PD-1_(pem)/anti-HER2 andanti-PD-1_(BJHM)/anti-HER2 all have high affinity for PD-1 and HER2, andthe antigen affinity activity of bivalent monoclonal antibody wasrelatively well maintained. Among them, anti-PD-1_(BJHM)/anti-HER2 hasstronger PD-1 affinity than anti-PD-1_(pem)/anti-HER2.

Example 7. Simultaneous Binding Activity of Anti-PD-1/Anti-HER2Heterodimeric Antibody Molecule to Dual Target Antigens

Simultaneous binding ability of the PD-1/HER2 heterodimeric antibody todual target antigens was determined on PD-1-overexpressing CHO/PD-1cells (GenScript, Cat. No. M00529) and HER2-overexpressing SK-BR-3 cellsby flow cytometry (FACS).

CHO/PD-1 cells were stained according to a PKH26 kit (Sigma, Cat. No.SLBH4568V) instructions. Briefly, CHO/PD-1 cells were collected, andwashed once with serum-free medium. CHO/PD-1 was prepared as a cellsuspension of 2×10⁷/mL using Diluent C in the PKH26 kit. PKH26 dye wasdiluted to 4 μM. Then, they were mixed at 1:1. The mixed suspension hada cell density of 1×10⁷/mL and PKH26 concentration of 2 μM, andincubated for 1 hour at room temperature, and then incubated with anequal volume of FBS for 1 minute, followed by terminating the staining.The suspension was centrifuged at 400 g for 10 minutes, and washed twicewith a complete medium, and resuspended in the complete medium for lateruse. SK-BR-3 cells were stained according to a CFSE kit (Lifetechnology, Cat. No. C34554) instructions. Briefly, CFSE was dilutedwith PBS to a working concentration of 0.5 μM and pre-warmed at 37° C.SK-BR-3 cells were collected by centrifugation at 1000 rpm for 5minutes, and resuspended in the pre-warmed CFSE working solution,followed by incubation at 37° C. for 15 minutes. SK-BR-3 cells werecollected by centrifugation at 1000 rpm for 5 minutes, and resuspendedin the complete medium, followed by incubation for 30 minutes. The cellswere washed once with the complete medium, and resuspended in thecomplete medium for later use.

The above stained cells were collected by centrifugation and washed oncewith cold PBS containing 2% FBS. The cells were resuspended in cold PBScontaining 2% FBS at a cell density of 5×10⁶/mL. SK-BR-3 and CHO/PD-1were mixed at 1:1, and then 100 μL thereof was taken from each flow tube(i.e., 2.5×10⁵ of SK-BR-3 and 2.5×10⁵ of CHO/PD-1). Then, 100 μL of theheterodimeric antibody samples diluted with cold PBS containing 2% FBS,a control group, and an isotype control (human immunoglobulin, JiangxiBoya Biopharmaceutical Co., Ltd., national drug approval No. S19993012)were added at a final concentration of 5 nM, respectively. The flow tubewas incubated on ice for 30 minutes, and washed twice with PBScontaining 2% FBS.

As a result, as shown in Table 1 and FIG. 12, simultaneous binding ofthe heterodimeric antibody to both the PD-1-overexpressing CHO/PD-1cells and HER2-overexpressing SK-BR-3 cells was observed, suggestingthat the PD-1/HER2 heterodimeric antibody is able to trigger a closeassociation between SK-BR-3 and CHO/PD-1 cells, which is the basis for Tcell-mediated tumor cell killing.

TABLE 1 Percentage of cells triggering close association Name of sample% associated cells Isotype control 1.89 HER2 monoclonal antibody(100 nM)1.39 PD-1 monoclonal antibody_(pem) (100 nM) 1.26 PD-1 monoclonalantibody_(BJHM) (100 nM) 1.43 PD-1 monoclonal antibody + HER2 1.45monoclonal antibody(100 nM) anti-PD-1_(pem)/anti-HER2 (100 nM) 39.71anti-PD-1_(BJHM)/anti-HER2 (0.1 nM) 8.15 anti-PD-1_(BJHM)/anti-HER2 (1nM) 32.79 anti-PD-1_(BJHM)/anti-HER2 (10 nM) 36.00anti-PD-1_(BJHM)/anti-HER2 (100 nM) 38.12

Example 8. Blocking Activity of Anti-PD-1/Anti-HER2 HeterodimericAntibody Molecule Against Binding of PD-1 to PD-L1 or PD-L2 Ligand

Recombinant human PD-1-Fc was coated on a 96-well high adsorption ELISAplate using a phosphate buffer solution of pH 9.6 at a coatingconcentration of 1 μg/mL and a coating amount of 100 μL per well, andthe coating was performed at 4° C. overnight. The plate was washed withPBST five times. The plate was blocked with 300 μL/well of PBSTcontaining 1% BSA and incubated for 1 hour at 25° C., and washed withPBST five times. A heterodimeric antibody sample and a control eachserially diluted with PBST containing 1% BSA were added, andsimultaneously, biotin-labeled PD-L1-Fc at a final concentration of 1μg/mL or biotin-labeled PD-L2 at a final concentration of 4 μg/mL wasadded in an amount of 100 μL per well, and incubated at 25° C. for 1hour. The plate was washed with PBST five times. Then, horseradishperoxidase-labeled streptavidin (BD, Cat. No. 554066) diluted 1:1000with PBST containing 1% BSA was added in an amount of 100 μL per well,and incubated at 25° C. for 1 hour. The plate was washed with PBST fivetimes. A colorimetric substrate TMB was added in an amount of 100μL/well and developed for 10 minutes at room temperature. Colordevelopment was terminated by adding 100 μL/well of 1 M H₂SO₄. Theabsorbance at 450 nm was read on a microplate reader.

As a result, as shown in FIG. 13, anti-PD-1_(pem)/anti-HER2 andanti-PD-1_(BJHM)/anti-HER2 all have blocking activity against PD-1/PD-L1binding and PD-1/PD-L2 binding, and the blocking activity of bivalentmonoclonal antibody was relatively well maintained. Among them,anti-PD-1_(BJHM)/anti-HER2 has stronger PD-1 blocking activity thanPD-1_(pem)/anti-HER2.

Example 9. T Cell Cytokine Secretion-Enhancing Activity ofAnti-PD-1/Anti-HER2 Heterodimeric Antibody Molecule

Human PBMC cells (Lonza, Cat. No. CC-2702) were collected. Human PBMCcells were resuspended in a complete medium (RPMI 1640 containing 10%FBS) at a cell density of 2×10⁶/mL, and 100 μL/well (2×10⁵ cells perwell) of the cells were seeded in a 96-well plates. A PD-1/HER2heterodimeric antibody sample and a control each diluted with thecomplete medium were added in an amount of 50 μL per well. PHA (Sigma,Cat. No. L-2769) diluted with the complete medium at a finalconcentration of 1 μg/mL was added in an amount of 50 μL per well. Theplates were incubated in a carbon dioxide incubator at 37° C. After 3days of incubation, 50 μL of the supernatant was taken and used fordetection of cytokine IL-2 (Ray Biotech, Cat. No. ELH-IL2).

As shown in FIG. 14, human T cells activate IL-2 secretion under PHAstimulation. Addition of PD-1 antibody enhances T cell activation andpromotes cytokine secretion, while the PD-1/HER2 heterodimeric antibodyhas a similar effect as PD-1 monoclonal antibody, and promotes cytokineIL-2 secretion in a concentration-dependent manner.

Example 10. Pharmacokinetics of Anti-PD-1/Anti-HER2 HeterodimericAntibody Molecule in Rats

6-8 week-old female SD rats purchased from Beijing HuafukangBiotechnology were used as experimental materials. One week after therats were acclimated to the environment, they were randomized intogroups, each group containing 3 rats. Each group was administered oncewith PD-1 monoclonal antibody, HER2 monoclonal antibody, PD-1/HER2heterodimer antibody at a dose of 20 nmol/kg via intravenous route.Immediately after administration, 5 minutes, 30 minutes, 1 hour, 4hours, 8 hours, 24 hours, 48 hours, 72 hours, 96 hours, 120 hours, 168hours, 216 hours, 264 hours, 312 hours, 360 hours, 408 hours, and 480hours after administration, 0.2 mL to 0.3 mL of the blood was collectedfrom the eyelid. The blood samples were left at room temperature for 30minutes to 1 hour without using an anticoagulant, and after coagulation,the blood samples were centrifuged at 3000 rpm for 10 minutes. Theobtained serum samples were frozen and stored at −80° C. until use for atest.

The serum concentrations of PD-1 monoclonal antibody, HER2 monoclonalantibody, and PD-1/HER2 heterodimeric antibody were determined by ELISA.Briefly, human recombinant HER2 protein (Beijing Yiqiao Shenzhou, Cat.No. 10004-H08H) or recombinant PD-1 protein (Beijing Yiqiao Shenzhou,Cat. No. 10377-H08H) was coated on a high adsorption ELISA plateovernight using a carbonate buffer solution of pH 9.6 at 4° C. Theplates were washed with PBST. To prevent non-specific binding, theplates were blocked with PBST containing 5% skim milk and washed withPBST. Then, 10% mixed rat serum and the test serum sample diluted withPBST containing 1% BSA were added and incubated at 25° C. for 1 hour,and the plates were washed with PBST. Horseradish peroxidase-labeledanti-human IgG antibody (Chemicon, Cat. No. AP309P) diluted with PBSTcontaining 5% skim milk was added, and incubated at 25° C. for 1 hour.The plates were washed with PBST. Finally, color development was carriedout using a colorimetric substrate TMB for 10 minutes at roomtemperature. Color development was terminated by addition of 1 M H₂SO₄.The absorbance at 450 nm was read on a microplate reader.

As a result, as shown in FIG. 15, 20 nmol/kg of the PD-1/HER2heterodimeric antibody which was used in the single intravenousinjection showed good pharmacokinetic characteristics in rats. Thepharmacokinetic parameters of the anti-PD-1_(BJHM)/anti-HER2heterodimeric antibody are as follows: a half-life (t_(1/2)) was 207hours; area under the plasma drug concentration-time curve (AUClast) was33448 nM·hr; C₀ was 534 nM; apparent distribution volume (V_(d)) was 148mL/Kg; clearance (C_(L)) was 0.50 mL/hr/kg; and a mean residence time(MRT_(last)) was 159 hours.

1. A heterodimeric bispecific antibody comprising a firstantigen-binding functional domain capable of specifically binding toPD-1 and a second antigen-binding functional domain capable ofspecifically binding to HER2, wherein the heterodimeric bispecificantibody comprises a first Fc chain and a second Fc chain which arelinked to each other via one or more disulfide bonds, the first Fc chainand the second Fc chain being respectively linked to the PD-1antigen-binding functional domain and the HER2 antigen-bindingfunctional domain via a covalent bond or a linker, or the first Fc chainand the second Fc chain being respectively linked to the HER2antigen-binding functional domain and the PD-1 antigen-bindingfunctional domain via a covalent bond or a linker, and the first Fcchain and the second Fc chain comprise 5 amino acid substitutions at thefollowing positions: 1) amino acid substitutions at positions T366 andD399 of the first Fc chain and amino acid substitutions at positionsL351, Y407, and K409 of the second Fc chain; or 2) amino acidsubstitutions at positions T366 and K409 of the first Fc chain and aminoacid substitutions at positions L351, D399, and Y407 of the second Fcchain; wherein the first and second Fc chains comprising the above aminoacid substitutions have a tendency to undergo heterodimerization ratherthan homodimerization, and the amino acid positions are numberedaccording to the Kabat EU numbering system.
 2. The heterodimericbispecific antibody of claim 1, wherein the amino acid substitutions ofthe first Fc chain and the second Fc chain comprise: a) L351G, L351Y,L351V, L351P, L351D, L351E, L351K, or L351W; b) T366L, T366P, T366W, orT366V; c) D399C, D399N, D399I, D399G, D399R, D399T, or D399A; d) Y407L,Y407A, Y407P, Y407F, Y407T, or Y407H; e) K409C, K409P, K409S, K409F,K409V, K409Q, or K409R.
 3. The heterodimeric bispecific antibody ofclaim 1, wherein the amino acid substitutions comprise: a) T366L andD399R substitutions in the first Fc chain and L351E, Y407L, and K409Vsubstitutions in the second Fc chain; b) T366L and D399C substitutionsin the first Fc chain and L351G, Y407L, and K409C substitutions in thesecond Fc chain; c) T366L and D399C substitutions in the first Fc chainand L351Y, Y407A and K409P substitutions in the second Fc chain; d)T366P and D399N substitutions in the first Fc chain and L351V, Y407P,and K409S substitutions in the second Fc chain; e) T366W and D399Gsubstitutions in the first Fc chain and L351D, Y407P, and K409Ssubstitutions in the second Fc chain; f) T366P and D399I substitutionsin the first Fc chain and L351P, Y407F, and K409F substitutions in thesecond Fc chain; g) T366V and D399T substitutions in the first Fc chainand L351K, Y407T, and K409Q substitutions in the second Fc chain; h)T366L and D399A substitutions in the first Fc chain and L351W, Y407H,and K409R substitutions in the second Fc chain.
 4. The heterodimericbispecific antibody of claim 3, wherein the amino acid substitutionscomprise: a) T366L and K409V substitutions in the first Fc chain andL351E, Y407L, and D399R substitutions in the second Fc chain; b) T366Land K409C substitutions in the first Fc chain and L351G, Y407L, andD399C substitutions in the second Fc chain; c) T366L and K409Psubstitutions in the first Fc chain and L351Y, Y407A, and D399Csubstitutions in the second Fc chain; d) T366P and K409S substitutionsin the first Fc chain and L351V, Y407P, and D399N substitutions in thesecond Fc chain; e) T366W and K409S substitutions in the first Fc chainand L351D, Y407P, and D399G substitutions in the second Fc chain; f)T366P and K409F substitutions in the first Fc chain and L351P, Y407F,and D399I substitutions in the second Fc chain; g) T366V and K409Qsubstitutions in the first Fc chain and L351K, Y407T, and D399Tsubstitutions in the second Fc chain; h) T366L and K409R substitutionsin the first Fc chain and L351W, Y407H, and D399A substitutions in thesecond Fc chain.
 5. The heterodimeric bispecific antibody of claim 1,wherein the amino acid substitutions of the first Fc chain are T366L andD399R, and the amino acid substitutions of the second Fc chain areL351E, Y407L, and K409V.
 6. The heterodimeric bispecific antibody ofclaim 1, wherein the Fc chain is derived from IgG.
 7. The heterodimericbispecific antibody of claim 1, wherein the PD-1 and HER2antigen-binding functional domains are Fab fragments or scFv fragments.8. The heterodimeric bispecific antibody of claim 1, wherein the PD-1and HER2 antigen-binding functional domains are all Fab fragments. 9.The heterodimeric bispecific antibody of claim 1, wherein one of thePD-1 and HER2 antigen-binding functional domains is a Fab fragment andthe other is a scFv fragment.
 10. The heterodimeric bispecific antibodyof claim 7, wherein the Fab fragment comprises a first heavy chainvariable region and a second heavy chain variable region that aredifferent from each other and a first light chain variable region and asecond light chain variable region that are different from each other.11. The heterodimeric bispecific antibody of claim 1, wherein when thefirst Fc chain and the PD-1 antigen-binding functional domain linkedthereto and the second Fc chain and the HER2 antigen-binding functionaldomain linked thereto, or the first Fc chain and the HER2antigen-binding functional domain linked thereto and the second Fc chainand the PD-1 antigen-binding functional domain linked thereto arepresent alone or are present with a reducing agent, a weight ratio ofhomodimer formation is less than 50%.
 12. The heterodimeric bispecificantibody of claim 1, wherein an amino acid sequence of the bispecificantibody is selected from SEQ ID NOs. 2, 4, 6, 8, 10, 12, 14, 16, and18.
 13. An isolated polynucleotide encoding the heterodimeric bispecificantibody of claim
 1. 14. The isolated polynucleotide of claim 13, havinga sequence selected from SEQ ID NOs. 1, 3, 5, 7, 9, 11, 13, 15 and 17.15. A recombinant expression vector comprising the isolatedpolynucleotide of claim
 13. 16. The recombinant expression vector ofclaim 15, wherein the expression vector is a plasmid vector XOGCobtained by modifying pcDNA.
 17. A host cell comprising (a) the isolatedpolynucleotide of claim 13; (b) an isolated polynucleotide having asequence selected from SEQ ID NOs. 1, 3, 5, 7, 9, 11, 13, 15 and 17; (c)a recombinant expression vector comprising the isolated polynucleotide(a); and/or (d) a recombinant expression vector comprising the isolatedpolynucleotide (b).
 18. The host cell of claim 17, wherein the host cellis selected from a human embryonic kidney cell HEK293, or HEK293T,HEK293E, or HEK293F derived from the HEK293 cell; and a Chinese hamsterovary cell CHO, or CHO-S, CHO-dhfr⁻, CHO/DG44, or ExpiCHO derived fromthe CHO cell.
 19. A composition comprising (a) the heterodimericbispecific antibody of claim 1, (b) the isolated polynucleotide encodingthe heterodimeric bispecific antibody (a); (c) an isolatedpolynucleotide having a sequence selected from SEQ ID NOs. 1, 3, 5, 7,9, 11, 13, 15 and 17; (d) a recombinant expression vector comprising theisolated polynucleotide (b); (e) a recombinant expression vectorcomprising the isolated polynucleotide (c), (f) a host cell comprising(b), (c), (d), and/or (e), and a pharmaceutically acceptable carrier.20. A method of producing the heterodimeric bispecific antibody of claim1, the method comprising: 1) expressing any one of the following(a)-(d): (a) the isolated polynucleotide encoding the heterodimericbispecific antibody of claim 1; (b) an isolated polynucleotide having asequence selected from SEQ ID NOs. 1, 3, 5, 7, 9, 11, 13, 15 and 17; (c)a recombinant expression vector comprising the isolated polynucleotide(a); (d) a recombinant expression vector comprising the isolatedpolynucleotide (b) in a host cell; 2) purifying and reducing eachprotein expressed in the host cell; and 3) mixing the reduced proteinsto obtain a mixture and then oxidizing the mixture.
 21. The method ofclaim 20, wherein the host cell is selected from a human embryonickidney cell HEK293, or HEK293T, HEK293F, or HEK293E derived from theHEK293 cell; and a Chinese hamster ovary cell CHO, or CHO-S, CHO-dhfr⁻,CHO/DG44, or ExpiCHO derived from the CHO cell.
 22. The method of claim20, wherein the reducing comprises: 1) adding a reducing agent, whereinthe reducing agent is selected from 2-mercaptoethylamine,dithiothreitol, tri(2-carboxyethyl)phosphine, other chemicalderivatives, and a combination thereof, 2) performing a reductionreaction in the presence of dithiothreitol at a concentration of 0.1 mMor more at 4° C. for 3 hours, and 3) removing the reducing agent bydesalting, etc.
 23. The method of claim 20, wherein the oxidizingcomprises: 1) oxidizing in air or adding an oxidizing agent, wherein theoxidizing agent is selected from L-dehydroascorbic acid and otherchemical derivatives, and 2) performing an oxidization reaction in thepresence of L-dehydroascorbic acid at a concentration of 0.5 mM or moreat 4° C. for 5 hours.
 24. The method of claim 20, further comprisingisolating and purifying.
 25. (canceled)
 26. (canceled)
 27. A method ofpreventing and/or treating a disease, the method comprisingadministering one or more selected from the group consisting of (a)-(g):(a) the heterodimeric bispecific antibody of claim 1, (b) the isolatedpolynucleotide encoding the heterodimeric bispecific antibody (a); (c)an isolated polynucleotide having a sequence selected from SEQ ID NOs.1, 3, 5, 7, 9, 11, 13, 15 and 17; (d) a recombinant expression vectorcomprising the isolated polynucleotide (b); (e) a recombinant expressionvector comprising the isolated polynucleotide (c), (f) a host cellcomprising (b), (c), (d), and/or (e), and (g) a composition comprising(a), (b), (c), (d), (e), and/of (f) and a pharmaceutically acceptablecarrier, to a subject in need thereof.
 28. The method of claim 27,wherein the subject is a mammal.
 29. The method of claim 27, wherein thedisease is selected from the group consisting of leukemia, lymphoma,myeloma, brain tumor, head and neck squamous cell carcinoma, non-smallcell lung cancer, nasopharyngeal carcinoma, esophageal cancer, stomachcancer, pancreatic cancer, gallbladder cancer, liver cancer, coloncancer, breast cancer, ovarian cancer, cervical cancer, endometrialcancer, uterine sarcoma, prostate cancer, bladder cancer, renal cellcarcinoma, and melanoma.